Tandem Femto- and Nanomolar Analysis of Two Protein Biomarkers in Plasma on a Single Mixed Antibody Monolayer Surface Using Surface Plasmon Resonance.

Abstract

The multiplexed detection of protein biomarkers in plasma present over a range of clinically relevant concentrations continues to be difficult for surface-based bioaffinity detection platforms such as surface plasmon resonance (SPR). As well as nonspecific adsorption, challenges include quantitative comparison between targets whose concentrations differ by orders of magnitude, regenerating SPR chips after plasma exposure, and the two- or four-channel limitation of many commercial SPR instruments limiting sample throughput. In this article, we explore an approach where two protein biomarkers alpha-1 antitrypsin (AAT) and Tau 381 are detected in tandem within a single SPR channel at micromolar and femtomolar concentrations, respectively. This was achieved by creating a mixed antibody (antiAAT and antiTau) monolayer on the chip surface. After the adsorption of AAT and/or Tau, further specificity was obtained via the adsorption of a DNA aptamer specific to each target. The detection range for each target was controlled via the relative surface density ratio of each antibody type as well as each aptamer concentration. Calibration measurements were performed in both buffer and spiked plasma with the detection of native concentrations of ∼39 fM (Tau) and ∼65 μM (AAT) in a human plasma sample. Finally, tandem measurements of both targets within the same SPR signal channel were demonstrated at these very different concentrations.